Human epidermal growth factor receptor 2 (HER2; human HER2 has the UniProtKB/Swiss-Prot number P04626) also known as ErbB2 is a protein that in humans is encoded by the ERBB2 gene. Amplification or over-expression of this gene has been shown to play an important role in the pathogenesis and progression of certain types of cancer and in recent years it has evolved to become an important biomarker and target of disease therapy. HER2 is a trans-membrane receptor tyrosine kinase (RTK) belonging to the wider family of ErbB receptors (Bublil, E. M. and Yarden, Y. Curr. Opin. Cell Biol. 19(2), 124-34, 2007). The ErbB receptor family is conserved across vertebrates and also includes the family founder ErbB1 (also named epidermal growth factor receptor (EGFR) or HER1; P00533 number in UniProKB/Swiss-Prot for the human protein) and the more recently identified receptors HER3 (also named ErbB3; P21860 number in UniProKB/Swiss-Prot for the human protein) and HER4 (also named ErbB4; Q15303 number in UniProKB/Swiss-Prot for the human protein). All ErbB receptors share extensive sequence and domain homologies, and form functional homodimers (e.g. ErbB1-ErbB1, HER2-HER2 and HER4-HER4) and heterodimers in all combinations. Receptor homo- and heterodimerization occurs upon ligand binding or receptor overexpression, and in turn activates intracellular receptor kinase domains by autophosphorylation. This then triggers downstream intracellular signaling and biological responses. In contrast to the other ErbB-receptors, HER2 does not have any known ligand and is able to dimerize, which is strongly pronounced after its overexpression and is thereby activated without previous ligand binding. Importantly, HER3 has no active intracellular kinase domain and is activated through heterodimerization with other ErbB receptor family members leading to very potent downstream signaling. Such heterodimerization and activation of HER3 occurs upon ligand binding to HER3 or if a partnering receptor, such as HER2, is strongly overexpressed.
HER2 as well as all the other ErbB receptor family members are composed of four extracellular domains, which are sequentially named I, II, III and IV; where domain IV is the closest to the extracellular cell membrane and domain I the most distal. In ligand-deprived conditions, domains I and III in ErbB receptors share an intramolecular interaction that occludes domain II. This prevents receptor homo-/heterodimerization and signaling, since interaction between domains II of two neighboring ErbB receptors is required for dimerization (Burguess A. W., et al., Mol. Cell 12(3), 541-552, 2003). Ligand binding disrupts the interaction between domains I and III, which then causes a tethered-to-extended receptor conformational change and leaves domain II exposed. This makes the receptor promiscuous to dimerize with other extended ErbB receptors and initiate signaling. Interestingly, HER2 is the only ErbB receptor family member that is constitutively found in an extended conformation; hence domain II is continuously exposed and accessible for homo- and heterodimerization.
ErbB receptor dimerization and autophosphorylation leads to the activation of a plethora of key downstream signaling molecules involved in normal physiology as well as in disease. The nature of such activated signaling molecules depends to some extend on the composition of the active ErbB receptor dimers. For instance, HER1-HER1 and HER2-HER2 homodimers preferentially activate downstream extracellular-signal-regulated kinase (ERK) signaling and proliferation, whereas HER2-HER3 heterodimers also activate the PI3K-signaling pathway (including activation of the downstream kinase AKT) and thereby cell survival. In fact, AKT activation by HER2-HER3 signaling in tumor cells promotes survival and makes tumor cells resistant to HER2 targeting drugs, such as the monoclonal antibody trastuzumab (Berns K. et al., Cancer Cell 12, 395-402, 2007). Interestingly, inhibition of HER2-HER3 mediated PI3K-AKT signaling in these cells becomes rate-limiting and results in cell death. Apart from cell proliferation and survival, HER2 signaling has been also causally involved in other processes such as angiogenesis and migration.
HER2 is overexpressed in approximately 20% of all breast cancers. Due to its clinical relevance, HER2 became the first RTK against which a targeted biological was developed, namely trastuzumab (Herceptin®; Genentech). This antibody binds to domain IV of HER2 and inhibits HER2 signaling by several mechanisms that are not yet completely understood. These include induction of receptor internalization in tumor cells, which results in reduced HER2 expression levels and signaling and leads to an attenuated tumorigenic phenotype. Trastuzumab has changed the life of tens of thousands of breast cancer women, expanding their lifetime and quality of life. However, trastuzumab has mainly an anti-proliferative effect and tumors may escape from such treatment in advanced disease stages. In an attempt to develop more efficacious treatments, a new antibody was generated that recognized domain II or HER2, namely pertuzumab (Omnitarg®, Perjeta®; Genentech). In contrast to trastuzumab, this antibody was not developed to reduce the membrane expression levels of HER2, but to interfere with HER2 homo- and heterodimer formation by binding to and occluding the dimerization domain II of the receptor. Pertuzumab treatment has an unexpected low therapeutic efficacy in vitro and in vivo as single agent; nevertheless, its combination with trastuzumab shows synergistic effects. Therefore, the combination of both antibodies may become a standard of care therapy for breast cancer patients (Capelan M., et al., Ann. Oncol., 24, 273-82, 2013).
The preclinical and clinical success of the combination of trastuzumab and pertuzumab has led to the concept that dual targeting of domains II and IV in HER2 is required for superior anti-tumor efficacy. This is aligned with other molecules more recently generated to simultaneously target HER2 on domains II and IV. For instance, the Danish company Symphogen is developing antibody mixes against domains II and IV of HER2 that have shown some higher efficacy (i.e. superior to trastuzumab alone) in preclinical mouse tumor models.
Similarly, US2011/033460 describes that the combination of antibodies that bind domain I and domain IV of HER2 exhibits synergistic effects on DNA synthesis and viability of BT474 cells. Furthermore, US2011/033460 also describes bispecific antibodies that bind two different epitopes of HER2, one epitope located on domain I of HER2 and the other epitope located on domain IV of HER2.
WO 2009/068625 covers the development of biparatopic antibody constructs comprising a first antibody domain, which competes with trastuzumab for binding to HER2, and a second antibody domain, which binds to a different epitope or part of HER2. Interestingly, some constructs had an antagonistic effect of SKBR3 cell proliferation, whereas others had an agonistic effect. Especially, WO 2009/068625 covers the development of biparatopic antibody constructs comprising a first antibody domain, which competes with trastuzumab for binding to HER2 (i.e. binding domain IV of Her2) and a second antibody domain, which competes with pertuzumab for binding to HER2 (i.e. binding domain II of HER2). Constructs where the domain IV binding antibody domain was cloned N-terminally to the domain II binding antibody domain showed blocking of map kinase activation, whereas such a blocking was not observed with the other orientation (i.e., having the domain II binding antibody domain at the N-terminus). Overall, WO 2009/068625 describes a variety of biparatopic antibody constructs targeting HER2, which have to variable extends effects on SKBR3 cell proliferation (agonistic or antagonistic) or cell signaling, but no cytotoxic nor apoptotic effects were described.
Bivalent binding proteins, such as bivalent diabody molecules or bivalent affibodies targeting HER2, are described also (Nielsen, U. B., et al., Cancer Res., 60, 6434-6440, 2000; Steffen, A-C., Cancer Biother. Radiopharmaceut. 20, 239-248, 2005). Such molecules combine two times the same binding domain and thus are different to biparatopic molecules that comprise two binding domains each of which binds to a different epitope on the same target molecule.
As an alternative to antibody-derived therapeutics and SMIs, there are novel binding proteins or binding domains that can be used to specifically bind a target molecule (e.g. Binz, H. K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005) and thereby act as an antagonist. One such novel class of binding proteins or binding domains not possessing an Fc are based on designed repeat proteins or designed repeat domains (WO 2002/020565; Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, M. T., Binz, H. K and Amstutz, P., Drug Discov. Today 13, 695-701, 2008).
WO 2002/020565 describes how large libraries of repeat proteins can be constructed and their general application. Such designed repeat domains harness the modular nature of repeat proteins and may possess N-terminal and C-terminal capping modules to prevent the designed repeat domains from aggregation by shielding the hydrophobic core of the domain (Forrer, P., Stumpp, M. T., Binz, H. K. and Plückthun, A., FEBS letters 539, 2-6, 2003). This novel class of binding proteins includes designed ankyrin repeat proteins (DARPins). The generation of monospecific DARPins binding to HER2 were previously described (e.g. Steiner, D., Forrer, P. and Plückthun, A., J. Mol. Biol. 382, 1211-1227, 2008; Zahnd, C., Pecorari, F., Straumann, N., Wyler, E. and Plückthun, A., J. Biol. Chem. 281(46), 35167-35175, 2006).
Recently, a bispecific designed ankyrin repeat protein was described, which targets HER2 (Jost, Ch., et al., Structure 21, 1-13, 2013). The authors show that binding of two ankyrin repeat domains connected by a short linker (longer linkers do not work as well), one targeting domain I of Her2 and the other domain IV of Her2, causes stronger cytotoxic effects on BT474 cells as compared to trastuzumab alone, which targets domain IV of Her2. This biparatopic repeat protein works by intra-molecular cross-linking of two Her2 molecules; i.e., it connects two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to the observed cytotoxic effects.
Even though the prior art indicates that targeting of HER2 is beneficial for the therapy of diseases, such as cancer, there is a clear need to generate binding proteins targeting HER2 with higher efficacy.